Comprehensive Stool DNA/RNA Extraction

DNA and RNA extractions were performed at the Liggins Institute (University of Auckland, New Zealand) using a modified protocol of the AllPrep DNA/RNA Mini Kit (#80204, Qiagen, Germany) as described previously^{19 (link)}. Briefly, stool aliquots were thawed and incubated in 100 µl of lysis buffer (30 mM Tris–HCl, 1 mM EDTA, 15 mg/ml lysozyme) and 10 µl proteinase K for 10 min at room temperature with regular agitation. Samples were then mixed with 1.2 ml RLT plus buffer (Qiagen, Germany), 12 µl beta-mercaptoethanol, and 1 ml of acid washed glass beads (≤ 106 µm, − 140 U.S. sieve; #G4649-100G, Sigma Aldrich, USA) and shaken vigorously at 30 Hz for 10 min on a TissueLyser II (Qiagen, Germany). The homogenate was then passed through a QIAshredder spin column (#79654, Qiagen, Germany), before continuing with the standard AllPrep DNA/RNA Mini Kit protocol (#80204, Qiagen, Germany). DNA was eluted in 75 µl of EB buffer and RNA eluted in 50 µl RNase free water. DNA was quantified by Qubit dsDNA High Sensitivity Assay Kit (#Q32851, Invitrogen, California, USA). RNA was quantified by Qubit RNA High Sensitivity Assay Kit (#Q32852, Invitrogen, California, USA) and quality assessed using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer instrument (Agilent technologies, California, USA). Sample data (including RNA integrity Number, RIN) is available in Table S1.